The present invention relates to a permanent amniocytic cell line comprising at least one nucleic acid which brings about expression of the gene products of the adenovirus E1A and E1B regions. The present invention further relates to the production of a permanent amniocytic cell line and to its use for producing gene transfer vectors and/or adenovirus mutants. Further aspects are the use of amniocytes and of the adenoviral gene products of the E1A and E1B regions for producing permanent amniocytic cell lines.
Adenoviruses
Adenoviruses are a relatively homogeneous group of viruses characterized by an icosahedral capsid which consists mainly of the virally encoded hexon, penton and fiber proteins, and of a linear, double-stranded DNA genome with a size of about 36 kilobases (kb). The viral genome contains at the ends the inverted terminal repeat sequences (ITRs) which comprise the viral origin of replication. There is furthermore at the left-hand end of the genome the packaging signal which is necessary for packaging of the viral genome into the virus capsids during an infection cycle. Adenoviruses have been isolated from many species. There are more than 40 different human serotypes based on parameters which discriminate between the various serotypes, such as hemagglutination, tumorigenicity and DNA sequence homology (Wigand et al., in: Adenovirus DNA, Doerfler ed., Martinus Nijoff Publishing, Boston, pp. 408-441, 1986). Adenoviral vectors to date are usually derived from serotypes 2 (Ad2) and 5 (Ad5). Infections by Ad2 and Ad5 are endemic in humans. Ad2 and Ad5 are not oncogenic in humans and have good safety documentation because vaccinations have been performed on military personnel successfully and without complications in the USA (Pierce et al., Am. J. Epidemiol. 87, 237-246, 1968). The biology of adenoviruses is relatively well understood because adenoviruses have played an essential part in molecular biology as experimental tool for elucidating various fundamental biological principles such as DNA replication, transcription, RNA splicing and cellular transformation. Adenoviral particles enter the cell during an infection through receptor-mediated endocytosis in which, according to the current view, interaction of the knob domain of the fiber protein with the coxsackie adenovirus receptor (CAR) mediates adhesion of the virus particle to the cell surface (Bergelson et al., Science 275, 1320-1323, 1997). In a second step there is internalization of the virus particle, for which interaction of the penton base with integrins plays an essential part (Wickham et al., Cell 73, 309-319, 1993). After the particle has entered the cell, the viral genome gets into the cell nucleus as DNA-protein complex. The adenoviral infection cycle is divided into an early and a late phase which are separated by the start of adenoviral replication (Shenk, in: Virology, Fields ed., Lippincott-Raven Publishing, Philadelphia, pp. 2111-2148, 1996). In the early phase there is expression of the early viral functions E1, E2, E3 and E4. The late phase is characterized by transcription of late genes which are responsible for the expression of viral structural proteins and for the production of new viral particles.
E1A is the first viral gene to be expressed by the viral chromosome after the cell nucleus is reached. The E1A gene codes for the 12S and 13S proteins which are formed by alternative splicing of the E1A RNA. The E1A proteins activate the transcription of a number of cellular and viral genes by interacting with transcription factors. The main functions of E1A are a) activation of the other early viral functions E1B, E2, E3 and E4 and b) inducing resting cells to enter the S phase of the cell cycle. Expression of E1A on its own leads to programmed cell death (apoptosis).
E1B is one of the early viral genes activated by E1A. The E1B gene codes for the E1B 55 kD protein and the E1B 19 kD protein, which result through alternative splicing of the E1B RNA. The 55 kD protein modulates the progression of the cell cycle by interacting with the p53 tumor suppressor gene, is involved in preventing the transport of cellular mRNA in the late phase of the infection, and prevents E1A-induced apoptosis of cells. The E1A 19 kD protein is likewise important for preventing E1A-induced apoptosis of cells.
All human adenoviruses are able to transform rodent cells in cell culture. As a rule, coexpression of E1A and E1B is necessry for oncogenic transformation.
The protein IX gene which codes for a structural component of the viral capsid is embedded in the E1B transcription unit.
The E2A and E2B genes code for various proteins which are essential for replication of the viral genome. These comprise the precursor protein of the terminal protein (pTP), the DNA polymerase (Pol) and the single strand-binding protein (SSBP). On replication, pTP binds to the ITRs of the viral genome. There it acts as protein primer for DNA replication, which is initiated by Pol together with cellular factors. Pol, SSBP and the cellular factor NFII, and presumably other factors, are necessary for DNA chain extension.
E4 codes for various proteins. Inter alia, the E4 34 kD protein blocks, together with the E1B 55 kD protein, the accumulation of cellular mRNAs in the cytoplasm, and at the same time it facilitates the transport of viral RNAs from the cell nucleus into the cytoplasm.
After the start of replication of the viral genome there is expression of viral structural proteins which are necessary for establishment of the viral capsid and for complexation of the viral DNA with virally encoded DNA-binding proteins. There is evidently initial formation of an empty capsid, into which the viral genome subsequently enters. A cis element on the viral genome is necessary for this process, the so-called packaging signal which is located at the left-hand end of the viral genome and, in the case of Ad5, extends over a region from base pair 260 to base pair 460 (Hearing et al., J. Virol. 62, 2555-2558, 1987; Graeble and Hearing, J. Virol. 64, 2047-2056, 1990). The packaging signal overlaps with the E1A enhancer which is essential for activity of the E1A promoter. The exact mechanism of the packaging of the viral genome into the virus capsids is not clear but it is probable that interaction of cellular and/or viral proteins with the packaging signal is necessary for this.
Adenovirus Vectors
Adenoviral vectors are particularly important as expression vectors, especially for the purpose of gene therapy. There are several reasons for this: the biology of adenoviruses has been thoroughly investigated. The virus particles are stable and can be produced relatively simply and in high titers. Genetic manipulation of the adenoviral genome is easy. Adenovirus vectors are able efficiently to transduce replicating and nonreplicating cells in vitro and in vi vo.
a) First-generation Adenoviral Vectors
First-generation adenoviral vectors (Gilardi et al., FEBS Letters 267, 60-62, 1990; Stratford-Perricaudet et al., Hum. Gene Ther. 1, 241-256, 1990) are characterized by deletions of the E1A and E1B genes. E1A and E1B have transforming and transactivating properties. In addition, E1A is necessary for activating viral genes and E1B is necessary for the accumulation of viral transcripts. In some vectors in addition E3 is deleted in order to increase the capacity for uptake of foreign DNA. E3 is dispensable for producing adenoviruses in cell culture. The capacity for uptake of foreign DNA is about 8 kb. First-generation adenovirus vectors have to date been produced mainly in 293 cells (see below) which complement the E1A and E1B deficit of the vectors.
b) Second-generation Adenoviral Vectors
Second-generation adenoviral vectors are characterized by deletions of E2 and/or E4 in addition to deletions of E1A and E1B (Engelhardt et al., Proc. Natl. Acad. Sci., USA 91, 6196-6200, 1994; Yang et al., Nature Genet., 7, 362-367, 1994; Gorziglia et al., J. Virol. 70, 4173-4178, 1996; Krougliak and Graham, Hum. Gene Ther. 6, 1575-1586, 1995; Zhou et al., J. Virol. 70, 7030-7038, 1996). In some vectors in addition E3 is deleted in order to increase the capacity for uptake of foreign DNA. Second-generation adenoviral vectors were developed in order to reduce further the transcription of viral genes and the expression of viral proteins and in order thus to diminish further the antiviral immune response. The capacity for uptake of foreign DNA is negligibly increased by comparison with first-generation adenoviral vectors. Second-generation adenovirus vectors are produced in cell lines which, in addition to E1A and E1B, complement the particular deficit (E2 and/or E4).
c) Adenoviral Vectors of Large DNA Capacity
Adenoviral vectors of large DNA capacity are characterized by containing no viral coding DNA sequences (Kochanek et al., Proc. Natl. Acad. Sci. U.S.A. 93, 5731-5736, 1996; Fisher et al., Virology 217, 11-22, 1996; Kumar-Singh and Chamberlain, Hum. Mol. Genet. 5, 913-921, 1996). These vectors only contain the viral ends with inclusion of the ITRs and of the packaging signal. The capacity for uptake of foreign DNA is about 37 kb because by far the major part of the adenoviral genome is deleted. Various systems have been described for producing adenoviral vectors of large DNA capacity (Kochanek et al., supra; Parks et al., Proc. Natl. Acad. Sci. USA 93, 13565-13570, 1996; Hardy et al., J. Virol. 71, 1842-1849, 1997). The advantage of these adenoviral vectors with large DNA capacity compared with first- and second-generation adenoviral vectors is the larger capacity for uptake of foreign DNA and a lower toxicity and immunogenicity (Schiedner et al., Nature Genet. 18, 180-183, 1998; Morral et al., Hum. Gene Ther. 9, 2709-2716, 1998). Currently, adenoviral vectors of large capacity are produced with the aid of an E1A- and E1B-deleted helper virus which provides the viral functions necessary for a productive infection cycle in trans. To date, adenoviral vectors of large DNA capacity have been produced in 293 cells or in cell lines derived from 293 cells. In one of the production methods (Parks et al., supra; Hardy et al., supra), adenoviral vectors are produced in modified 293 cells which, in addition to E1A and E1B, express the Cre recombinase of bacteriophage P1. In this system, the packaging signal of the helper virus is flanked by loxP recognition sequences of bacteriophage P1. On infection of Cre-expressing 293 cells with helper virus and the adenoviral vector of large DNA capacity, the packaging signal of the helper virus is excised. For this reason there is packaging mainly of the vector containing a normal packaging signal but not of the helper virus.
d) Deleted Adenoviral Vectors
These vectors have been described as first-generation vectors which have the loxp recognition sequences of bacteriophage P1 positioned in the viral genome in such a way that, on infection of Cre-expressing 293 cells, most of the viral coding sequences or all the viral coding sequences are deleted by recombination between the loxP recognition sequences. The size of the genome of these vectors is about 9 kb. The capacity for uptake of foreign DNA is likewise about 9 kb (Lieber et al., J. Virol. 70, 8944-8960, 1996).
Adeno-associated Virus (AAV)
AAV belongs to the family of parvoviruses, genus dependovirus, and has two different life forms, occurring either as lytic virus or as provirus. For a lytic infection to take place the virus requires coinfection with a helper virus (adenovirus, vacciniavirus, herpes simplex virus). In the absence of a helper virus, AAV is unable to replicate, integrates into the genome and exists there as inactive provirus. When cells harboring AAV as integrated provirus are infected, for example with adenovirus, the provirus is able to enter a lytic infection cycle again (Samulski, Curr. Opin. Genet. Dev. 3, 74-80, 1993).
AAV capsids contain a single-stranded, linear DNA genome with either positive or negative polarity. Several AAV serotypes exist. The serotype which has been investigated most is AAV-2. The genome of AAV-2 consists of 4680 nucleotides. The genome contains at the ends inverted terminal repeat sequences (ITRs) having a length of 145 base pairs. The first 125 base pairs form a T-shaped hairpin structure consisting of two internal palindromes.
The AAV genome codes for nonstructural replication (Rep) proteins and for structural capsid (Cap) proteins. The various replication proteins (Rep78, Rep68, Rep52, Rep40) are generated by using different promoters (p5 and p19) and by alternative splicing. The various capsid proteins (VP1, VP2, VP3) are generated by alternative splicing using the p40 promoter.
AAV Vectors
AAV vectors contain only the ITRs of AAV and some adjacent, noncoding AAV sequences. For this reason, the capacity for uptake of foreign DNA is about 4.5 kb. Various systems have been described for producing recombinant AAV vectors (Skulimowski and Samulski, in: Methods in Molecular Genetics, Vol. 7, Adoph ed., Academic Press, pp. 3-12). The components necessary for replication, expression and packaging of the recombinant vector are provided in these systems. Specifically, these are expression cassettes which code for the Rep and Cap proteins of AAV, and the adenoviral helper functions. The adenoviral helper functions necessary for AAV production are, specifically, E1A, E1B, E2, E4 and VA. The E1A and E1B functions are provided in the 293 cells which have been used for production to date. In the production processes described to date, the E2, E4 and VA functions are currently usually provided either by coinfection with adenovirus or by cotransfection with E2-, E4- and VA-expressing plasmids (Samulski et al., J. Virol. 63, 3822-3828, 1989; Allen et al., J. Virol. 71, 6816-6822, 1997; Tamayose et al., Hum. Gene Ther. 7, 507-513, 1996; Flotte et al., Gene Ther. 2, 29-37, 1995; Conway et al., J. Virol. 71, 8780-8789, 1997; Chiorini et al., Hum. Gene Ther. 6, 1531-1541, 1995; Ferrari et al., J. Virol. 70, 3227-3234, 1996; Salvetti et al., Hum. Gene Ther. 9, 695-706, 1998; Xiao et al., J. Virol. 72, 2224-2232, 1998, Grimm et al., Hum. Gene Ther. 9, 2745-2760, 1998; Zhang et al., Hum. Gene Ther. 10, 2527-2537, 1999). Alternatively, strategies have been developed in which adenovirus/AAV or herpes simplex virus/AAV hybrid vectors have been used to produce AAV vectors (Conway et al., supra; Johnston et al., Hum. Gene Ther. 8, 359-370, 1997, Thrasher et al., Gene Ther. 2, 481-485, 1995; Fisher et al., Hum. Gene Ther. 7, 2079-2087, 1996; Johnston et al., Hum. Gene Ther. 8, 359-370, 1997). It is common to all these processes that E1A- and E1B-expressing 293 cells are currently used for production.
Producer Cell Lines
For safety reasons, adenoviral vectors intended for use in humans usually have deletions of the E1A and E1B genes. Production takes place in complementing cell lines which provide the E1 functions in trans. Most adenoviral vectors to date have been produced in the 293 cell line. In recent years, further cell lines which can be used to produce E1-deleted adenoviral vectors have been produced.
a) HEK 293 Cells
HEK 293 cells were for a long time the only cells which could be used to produce E1-deleted adenoviral vectors. HEK 293 cells were produced in 1977 by transfection of sheared adenoviral DNA into human embryonic kidney cells (HEK cells). In a total of eight transfection experiments each with an average of 20 HEK cultures it was possible to obtain only a single immortalized cell clone (Graham et al., J. Gen. Virol. 36, 59-74, 1977). The cell line (HEK 293 cells) established from this cell clone contains the complete left-hand 11% of the adenoviral genome (base pair 1 to 4344 of the Ad5 genome), including the E1A and E1B genes and the left-hand ITR and the adenoviral packaging signal (Louis et al., Virology 233, 423-429, 1997). A considerable problem for the production of adenoviral vectors is the sequence homology between E1-deleted adenoviral vectors and the portion of adenoviral DNA integrated into 293 cells. Homologous recombination between the vector genome and the adenoviral DNA integrated into 293 cells is responsible for the generation of replication-competent adenoviruses (RCA) (Lochmxc3xcller et al., Hum. Gene Ther. 5, 1485-1491, 1994; Hehir et al., J. Virol. 70, 8459-8467, 1996). HEK 293 cells are for this reason unsuitable for producing adenoviral vectors of pharmaceutical quality because production units are often contaminated with unacceptable amounts of RCA. RCA is unacceptable in products produced for clinical use because replication-competent adenoviruses have a distinctly higher toxicity than replication-defective adenoviruses, are capable of uncontrolled replication in human tissues, and are moreover able to complement replication-defective adenoviruses (Lochmxc3xcller et al., supra; Imler et al., Hum. Gene Ther. 6, 711-721, 1995; Hehir et al., supra).
b) Human Embryonic Retinal Cells (HER Cells) and Established Cell Lines
Although rodent cells can easily be transformed with adenoviral E1 functions, primary human cells have proved to be relatively resistant to transformation with E1A and E1B. As mentioned above, Graham and coworkers were able to isolate only a single cell clone from HEK cells which had been transfected with sheared Ad5 DNA. Gallimore and coworkers attempted for a long time unsucessfully to transform primary HEK cells with E1 functions of Ad12 (Gallimore et al., Anticancer Res., 6, 499-508, 1986). These experiments were carried out unsuccessfully over a period of three years with more than 1 mg of the EcoRI cDNA fragment of Ad12 containing the E1A and E1B genes. After many attempts it was possible, despite a large number of experiments carried out, to isolate only four Ad12-E1 HEK cell lines (Whittaker et al., Mol. Cell. Biol., 4, 110-116, 1984). Likewise, Gallimore and coworkers attempted unsuccessfully to transform other primary human cells with E1 functions, including keratinocytes, skin fibroblasts, hepatocytes and urothelial cells (Gallimore et al., Anticancer Res., 6, 499-508, 1986). The only human cell type which it has been possible to date to transform reproducibly with adenoviral E1 functions comprises human embryonic retinal cells (HER cells). HER cells are a mixture of cells derived from the white neural retina. To obtain these cells it is necessary to remove the eye from the orbital cavity of a human fetus, normally between weeks 16 and 20 of gestation. The eye is opened with a horizontal incision and the white neural retina can be removed with forceps and placed in cell culture.
Based on earlier observations that a) Ad12-induced tumors are primarily derived from primitive neural epithelium (Mukai et al., Prog. Neuropathol. 3, 89-128, 1976) and that b) Ad12 induces retinal tumors in rats and baboons after intraocular inoculation (Mukai et al., supra; Mukai et al., Science 210, 1023-1025, 1980), Byrd and coworkers found that human embryonic retinoblasts (HER cells) can be transformed with the E1 genes of Ad12 (Byrd et al., Nature 298, 69-71, 1982). Although the efficiency of transformation of HER cells was less than that of primary rat cells, the efficiency of transformation was more than 100 times higher than that of HEK cells. The investigations were initiated in order to produce complementing cell lines which could be used to isolated Ad12 E1 mutants.
In further investigations by this research group (Gallimore et al., Cancer Cells 4, 339-348, 1986) it was shown that HER cells can be transformed efficiently with plasmid DNA which expresses the E1A and E1B genes of Ad5. The efficiency of transformation and the establishment of E1A- and E1B-expressing cell lines was about 20 times higher with the E1 genes of Ad5 than with E1 genes of Ad12.
Based on these data, Fallaux and coworkers (Fallaux et al., Hum. Gene Ther. 7, 215-222, 1996; Fallaux et al., Hum. Gene Ther. 9, 1909-1917, 1998) established E1A- and E1B-expressing cell lines by transforming HER cells with plasmids which expressed the E1A and E1B genes of Ad5. The cell line 911 was produced by transformation with a plasmid which contains the E1A and E1B genes of Ad5 (nucleotides 79-5789 of the Ad5 genome) and expresses E1A under the control of the natural E1A promoter (Fallaux et al., supra; Patent Application WO97/00326). It was possible to establish further E1A- and E1B-expressing HER cell lines by transfecting a plasmid which contains nucleotides 459-3510 of the Ad5 genome, in which the E1A gene is under the control of the human phosphoglycerate kinase (PGK) promoter, and in which the natural E1B polyadenylation signal is replaced by the poly(A) sequence of the heptatitis B virus (HBV) surface antigen (Fallaux et al., supra; Patent Application WO97/00326). These HER cell lines have been referred to as PER cell lines. The advantage of these newer PER cell lines compared with 293 cells or the 911 cell line is the lack of sequence homology between the DNA of first-generation adenoviral vectors and the integrated Ad5 DNA. For this reason there is a marked reduction in the possibility of the generation of RCA. These E1A- and E1B-transformed HER cell lines (911 cells and PER cells) were able to complement the E1 deficit of first-generation adenoviral vectors and thus be used to produce these vectors.
In a similar way, a cell line which was established by transforming HER cells with the plasmid pTG6559 is mentioned in a publication by Imler and coworkers (Imler et al., supra; see also WO 94/28152). The plasmid pTG6559 contains the coding sequences of the E1A and E1B genes and of the protein IX gene (nucleotides 505-4034 of the Ad5 genome), with the E1A gene being under the control of the mouse phosphoglycerate kinase (PGK) promoter, and the joint polyadenylation signal of the E1B and protein IX genes having been replaced by the polyadenylation signal of the rabbit xcex2-globin gene.
In contrast to the described attempts to establish primary human cells by transformation with the E1A and E1B genes of Ad5, attempts have been made in a few cases to express E1A and E1B of various serotypes stably in previously established cell lines (Grodzicker et al., Cell, 21, 453-463, 1980; Babiss et al., J. Virol. 46, 454-465, 1983; Shiroki et al., J. Virol. 45, 1074-1082, 1983; Imler et al., supra; see also WO 94/28152). The disadvantages of these cell lines are the need for coexpression of a selection marker and the frequently deficient stability of E1A and E1B expression. Since these cell lines are immortalized cell lines from the outset, expression of E1A and E1B is not necessary for survival of the cell lines, so that natural selection by E1A and E1B is unnecessary in this case and in contrast to the use of primary cells.
In the past, the production of cell lines for producing adenoviral vectors or for producing AAV vectors was associated with particular difficulties. Human embryonic kidney cells (HEK cells) can be obtained from the kidney of human fetuses. This is done by removing a kidney from a fetus and placing kidney cells in the cell culture. Transfection of HEK cells with sheared AdS DNA and integration of the left-hand end of the Ads DNA, and expression of the E1A and E1B genes resulted in transformation of the cells in a single published case. It was possible to establish a single cell line (293 cells) in this way (Graham et al., supra; see above xe2x80x9cProducer cell linesxe2x80x9d, section a). 293 cells are used to produce adenoviral vectors and to produce AAV vectors.
Human embryonic retinal cells (HER cells) can be obtained from the eyeball of human fetuses. This is done by removing an eye from the fetus and placing cells from the retina in culture. It was possible by transfecting HER cells with the adenoviral E1A and E1B genes to transform HER cells (see above xe2x80x9cProducer cell linesxe2x80x9d, section b). Cells transformed with E1A and E1B can be used to produce adenoviral vectors.
It is necessary in both cases to remove an organ from human fetuses, which are derived either from a spontaneous or therapeutic abortion or from a termination of pregnancy on social grounds, and to establish a cell culture from this organ. After establishment of a primary culture, these cells can then be transformed by transfection with the adenoviral E1A and E1B genes. Cell lines established in this way and expressing E1A and E1B can then be used to produce adenoviral vectors or AAV vectors.
It is evident that it is complicated to obtain primary cells from organs from fetuses. Since a primary culture can be established only from fresh tissue, special logistic efforts are needed to obtain suitable tissue. In addition, the use of fetal tissue derived either from a spontaneous abortion, a therapeutic abortion or from a termination of pregnancy on social grounds makes special ethical considerations and care necessary for establishment of a primary culture. Although the inventors"" laboratory is situated in a gynecology clinic where terminations of pregnancy are frequently performed, it was not possible to obtain suitable tissue over a period of more than one year. Removal of fetal tissue after abortion requires a declaration of consent by the pregnant woman after receiving appropriate information. It was frequently impossible to obtain the consent of the pregnant woman for the organ-removal intervention after she had received detailed information about the project, i.e. the removal of an eye from the fetus for scientific medical investigations.
The use of a permanent amniocytic cell line for producing gene transfer vectors has not previously been described. There have merely been a report of human amniocytes which have been transformed with the simian virus (SV40) and/or the Kirsten sarcoma virus (Sack, In Vitro 17 pp. 1-19, 1981; Walen, et al., In Vitro Cell Dev. Biol. 22, 57-65, 1986). Infection with SV40 alone conferred an extended lifetime (called immortalization), whereas infection with the Kirsten sarcoma virus alone did not extend the lifespan. Infection with both viruses finally led to a malignant tumor cell (Walen and Arnstein, supra). It should be noted in this connection that SV40-transformed amniocytic cell lines are unsuitable for producing gene transfer vectors because these cells themselves produce SV40, which is known to be an oncogenic virus (Graffney et al., Cancer Res. 30, 871-879, 1970). The transformability of human cells with SV40 moreover provides no information about the transformability with the E1 functions of adenovirus and the use thereof for the production of gene transfer vectors. For example, keratinocytes can be transformed with SV40 (see Sack, supra), but keratinocytes evidently cannot, just like skin fibroblasts and hepatocytes, be transformed with Ad12 (Gallimore et al., 1986, supra). In terms of the production of viral vectors, especially adenoviral vectors, in immortalized cells, moreover, it is not just the immortalizability with the particular immortalization functions which is important; so too are good infectability and a good productive course of infection. These properties cannot be predicted; the question of whether a particular cell type can be used for producing gene transfer vectors must be determined anew for each cell type.
An object of the present invention was herefore to provide a novel process for the efficient, simple and easily reproducible production of an amniocytic cell line, and the use thereof inter alia for producing adenoviral vectors, AAV vectors and retroviral or lentiviral vectors.
It has been found, entirely surprisingly, that transfection of cells of the amniotic fluid (amniocytes), which are routinely obtained by amniotic fluid biopsy (amniocentesis) for diagnostic reasons during prenatal diagnosis, with the adenoviral E1A and E1B genes led to a large number of permanent cell lines which expressed the E1A and E1B genes in a functionally active manner and which are suitable for producing gene transfer vectors.
One aspect of the present invention is therefore a permanent amniocytic cell line comprising at least one nucleic acid which brings about expression of the gene products of the adenovirus E1A and E1B regions. A xe2x80x9cpermanent cell linexe2x80x9d means according to the present invention that the corresponding cells have been genetically modified in some way so that they are able to continue growing permanently in cell culture. By contrast, xe2x80x9cprimary cellsxe2x80x9d mean cells which have been obtained by removal from an organism and subculturing and which have only a limited lifetime. A permanent amniocytic cell line for the purpose of the present invention can be obtained by the process proposed herein, which comprises the transfection of primary amniocytes with the E1 functions of adenovirus. The at least one nucleic acid which brings about expression of the adenovirus E1 gene products can be any suitable nucleic acid or nucleic acids which lead to stable expression of these gene products. It/they can be integrated into the genome of the cell, i.e. chromosomally, or be present outside the chromosome, for example as episomally replicating plasmid or minichromosome. Expression of the various gene products can moreover be brought about by one and the same nucleic acid molecule or, for example, different nucleic acid molecules. xe2x80x9cExpressionxe2x80x9d means in the state of the art the process of production of a gene product which is a specific protein which brings about a specific trait or a specific property, or of RNA forms which are not translated into proteins (for example antisense RNAs, tRNAs). Suitable possibilities for achieving the desired expression will be evident to the skilled worker in the light of the present description, in particular of the proposed process too. The novel amniocytic cell line is suitable not only for use for producing gene transfer vectors in general but also, in particular, for producing first-generation adenoviral vectors characterized by deletions of the E1A and E1B genes, which are complemented by the cell line.
The at least one nucleic acid also preferably brings about expression of the gene products of the adenovirus E2A, E2B and/or E4 regions and/or of Cre recombinase. This makes the cell line particularly suitable for producing second-generation adenoviral vectors which are characterized by deletions of E2 and/or E4 genes in addition to the deletions of the E1A and E1B genes. Expression of the Cre recombinase of bacteriophage P1 is particularly advantageous in the production of adenoviral vectors of large capacity with the aid of an E1A- and E1B-deleted helper virus (see also Parks et al., supra; Hardy et al., supra). Expression of the gene products of the E1A region is advantageously under the control of a constitutive promoter, preferably the phosphoglycerate kinase (PGK) promoter. It is advantageous for expression of the gene products of the E1B region if it is under the control of an adenoviral promoter, preferably the adenoviral E1B promoter. A possible alternative to this is to employ, for example, a cytomegalovirus (CMV) promoter. All the adenoviral gene products are preferably derived from an adenovirus of the same subgenus, for example of human adenovirus type 5 (Ad5). The permanent amniocytic cell line is normally a human cell line, because this is particularly suitable for producing gene transfer vectors derived from human viruses, such as, for example, a human adenovirus or a human AAV.
A possible alternative to this is a cell line from primates or other mammals such as, for example, bovines, which is particularly suitable for producing gene transfer vectors derived from viruses occurring and endemic in particular species. For example, permanent amniocytic cell lines obtained by transformation of amniocytes with the E1A and E1B genes of a bovine adenovirus are suitable for producing vectors derived from a bovine adenovirus.
Another aspect of the present invention is a process for producing a permanent amniocytic cell line, in particular an amniocytic cell line as defined above, which comprises the transfection of amniocytes with at least one nucleic acid which brings about expression of the adenoviral gene products of the E1A region and E1B region. The resulting cell clones can then be isolated further where appropriate and, if required, be cloned to obtain single cell lines. The term xe2x80x9ctransfectionxe2x80x9d means herein any process suitable for introducing said nucleic acid(s) into the cells. Examples which may be mentioned are electroporation, liposomal systems of any type and combinations of these processes. The term xe2x80x9camniocytesxe2x80x9d means herein in the wider sense all cells which are present in the amniotic fluid and can be obtained by amniotic fluid biopsy. They are derived either from the amnion or from fetal tissue which is in contact with the amniotic fluid. Three main classes of amniocytes have been described and are distinguished on the basis of morphological criteria: fibroblast-like cells (F cells), epitheloid cells (E cells) and amniotic fluid cells (AF cells) (Hohn et al., Pediat. Res. 8, 746-754, 1974). AF cells are the predominant cell type. In the narrow sense, therefore, xe2x80x9camniocytesxe2x80x9d mean herein amniocytes of the AF type. Primary amniocytes are preferably used. Cells referred to as xe2x80x9cprimaryxe2x80x9d cells are those which can be obtained by removal from an organism and subculturing and have only a limited lifetime, whereas so-called xe2x80x9cpermanentxe2x80x9d cell lines are able to continue to grow unrestrictedly. It is particularly preferred in this connection to use human primary amniocytes which lead to the production of human cell lines (see above). However, it is also possible to use primary amniocytes from primates and other mammalian species such as from bovines. It will also be evident to the skilled worker in the light of the present description that it is possible to use analogously cells which can be obtained from the amniotic membranes, for example by trypsinization, or by a chorionic villus biopsy, for producing corresponding permanent cell lines.
The at least one nucleic acid which brings about expression of the adenoviral E1 gene products can be genomic DNA, cDNA, synthetic DNA, RNA and mRNA. The nucleic acid is preferably used in the form of a DNA expression vector. Examples thereof are integrative vectors, bacterial plasmids, episomally replicated plasmids or minichromosomes. Preference is given to expression plasmids whose integration into the genome of the recipient cell is brought about by transfection. The term xe2x80x9cat least one nucleic acidxe2x80x9d expresses the fact that the elements which bring about the expression may be present either on one and the same nucleic acid or on different nucleic acids. For example, separate nucleic acids may be provided for expression of the gene products of the E1A, E1B, E2A, E2B and/or E4 regions and/or of Cre recombinase. It is also conceivable that the amniocytes to be transfected already express one of these gene products so that only the expression of the other gene product(s) needs to be brought about, or that the expression of one or more of these gene products is switched on merely by introducing suitable regulatory elements. Suitable techniques and processes for the production and, where appropriate, mutagenesis of nucleic acids and for gene expression and protein analysis are available to the skilled worker (see, for example, Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989); Glover, D. M., DNA cloning: A practical approach, vol. II: Expression Systems, IRL Press (1995); Ausubel et al., Short protocols in molecular biology, John Wiley and Sons (1999); Rees, A. R. et al., Protein engineering: A practical approach, IRL press (1993)). It is preferred for the gene product or gene products of the E1A region to be expressed under the control of a constitutive promoter, in particular the phosphoglycerate kinase (PGK) promoter, and for the gene products of the E1B region to be expressed under the control of an adenoviral promoter, in particular the adenoviral E1B promoter. In place of the adenoviral promoter it is also possible to use, for example, a cytomegalovirus (CMV) promoter.
In a particular embodiment, transfection of the amniocytes and/or of the resulting cell line additionally brings about expression of the resulting gene products of the adenovirus E2A and/or E2B and/or E4 regions and/or of Cre recombinase. All the possibilities discussed previously or otherwise disclosed in the prior art are available to the skilled worker in this connection. Concerning the individual genes, reference is made in addition to the following information: E2A: Genbank Acc. #M73260; Kruiyer et al., Nucl. Acids Res. 9, 4439-4457, 1981; Kruiyer et al., Nucl. Acids Res. 10, 4493-4500, 1982. E2B: Genbank Acc. #M73260; Dekker et al., Gene 27, 115-120, 1984; Shu et al., Virology 165, 348-356, 1988. E3: Genbank Acc. #M73620; Cladaras et al., Virology 140, 28-43, 1985. E4: Genbank Acc. #M73620 and D12587; Virtanen et al., J. Virol. 51, 822-831, 1984; Dix et al., J. Gen. Virol. 73, 2975-2976, 1992. The reading frames are in some cases known only for Ad2 and can then usually be assigned by comparison of sequences in the case of, for example, Ad5. Cre recombinase: Genbank Acc. #X03453; Sternberg et al., J. Mol. Biol. 187, 197-212, 1986.
The adenoviral gene products are preferably all derived from a particular adenoviral serotype, in particular from human adenovirus type 5 (Ad5). The particular adenoviral serotype which is the origin of the E1A and E1B genes used for transforming amniocytes is not critical for this invention. Examples of adenoviral serotypes which can be used in the present invention are known in the prior art and include more than 40 human serotypes, for example Ad12 (subgenus A), Ad3 and Ad7 (subgenus B), Ad2 and Ad5 (subgenus C), Ad8 (subgenus D), Ad4 (subgenus E), Ad40 (subgenus F) (Wigand et al., in: Adenovirus DNA, Doerfler, ed., Martinus Nijhoff Publishing, Boston, pp. 408-441, 1986). In a preferred embodiment of this invention, adenoviral vectors derived from subgenus C are produced by transforming amniocytes with E1A and E1B genes which are derived from an adenovirus of the same subgenus. For example, adenoviral vectors of serotype 2 or 5 are produced by transforming amniocytes with the E1A and E1B genes of serotype 2 or 5. Adenoviral vectors based on Ad12 are produced by transforming amniocytes with the E1A and E1B genes of Ad12 etc. To produce non-human adenoviral vectors, including of the well-known adenoviruses derived from cattle, sheep, pigs and other mammals, amniocytic cell lines are produced by transforming amniocytes of the particular species. This is usually necessary because adenoviral functions usually cannot be complemented efficiently beyond species boundaries.
In a particular embodiment of the invention, amniocytes obtained for diagnostic reasons within the scope of prenatal diagnosis by amniotic fluid biopsy and no longer used for diagnostic purposes were transfected with an expression plasmid which expressed the E1A and E1B genes of Ad5. This construct was, designed so that the E1A gene was under the control of the mouse phosphoglycerate kinase promoter, and the E1B gene was under the control of the natural adenoviral E1B promoter. The natural E1B splice acceptor site and the E1B polyadenylation sequence were replaced by the corresponding sequences of the SV40 virus. A few weeks after transfection with the plasmid DNA, a large number of cell clones was observed, and these were isolated, cloned, established as immortalized cell lines and analyzed. All the analyzed cell clones expressed the E1A and E1B proteins. It was shown, by infection with an E1-deleted, xcex2-gal-expressing adenoviral vector and subsequent staining, that all these cells could be infected. Infection experiments with E1-deleted first-generation adenoviral vectors revealed that the cell lines are suitable for producing adenoviral vectors. In these experiments, the cell lines were initially infected with a xcex2-gal-expressing first-generation adenoviral vector. After 48-72 hours, when the cells showed a cytopathic effect (CPE), the cells were harvested and the adenoviral vector was freed of cells by freezing and thawing three times. Part of the cell lysate was used to infect 293 cells, and xcex2-gal expression was detected histochemically about 24 hours after the gene transfer. It was possible to calculate directly from the number of xcex2-gal-positive cells the yield of the vector by production in the individual cell lines. Amniocytic cell lines can be obtained in this way without difficulty and reproducibly and are very suitable for producing gene transfer vectors. Some of the isolated cell lines allowed adenoviral vectors to be produced just as well as or better than 293 cells. As was to be expected, the cell lines showed differences in the production of recombinant adenovirus vector (see FIG. 4). The cell lines N52.E6 and N52.F4 were distinguished by a rapid growth and particularly good production of adenoviral vectors, beneficial properties for the use of these cell lines for producing gene transfer vectors.
The design of the E1A- and E1B-expressing expression plasmid used for transforming the amniocytes precludes the generation of replication-competent adenoviruses (RCA) by homologous recombination of an adenoviral vector or of an adenoviral helper virus with the DNA integrated into the transformed amniocytes, in contrast to 293 cells. As an alternative to this, the individual E1 functions can be introduced on various expression plasmids into the cells to be transfected. It is, of course, also possible, as for the 293 cell line, to carry out a transformation of amniocytes and to test the batches generated in the production of gene transfer vectors for the RCA content, for example using a PCR or infection assay. The RCA-containing batches can then be discarded where appropriate.
Thus a further aspect of the present invention relates to a permanent amniocytic cell line which can be obtained by the process proposed herein. In a specific embodiment, the invention relates to the permanent amniocytic cell line N52.E6 which was deposited on Oct. 26, 1999 at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) in accordance with the Budapest treaty and has received the accession number DSM ACC2416.
In a further aspect, the present invention relates to the use of amniocytes for producing adenovirus-transformed permanent amniocytic cell lines. The term xe2x80x9cadenovirus-transformedxe2x80x9d means herein transformation by one or more transforming adenovirus genes. xe2x80x9cTransformationxe2x80x9d refers in this connection to conversion of a eukaryotic cell which is subject to growth control into a so-called permanent cell line which grows unrestrictedly. A further aspect is the use of the adenoviral gene products of the E1A and E1B regions for producing permanent amniocytic cell lines.
The present invention further comprises the use of a permanent amniocytic cell line for producing gene transfer vectors. xe2x80x9cGene transfer vectorsxe2x80x9d mean herein generally all vectors with which one or more therapeutic genes can be transferred or introduced into the desired target cells and, in particular, viral vectors having this property. In addition, the permanent amniocytic cell lines can be used to produce adenovirus mutants. xe2x80x9cAdenovirus mutantsxe2x80x9d mean adenoviruses which have at least one mutation in the E1A and/or E1B genes. In a preferred embodiment, they do not, however, in contrast to adenoviral gene transfer vectors, harbor any therapeutic genes. A typical example thereof comprises adenovirus mutants in which the E1B 55 kD protein is not expressed (for example the adenovirus mutant dl1520 (Barker et al., Virology 156, 107-121, 1987)). Adenovirus mutants in which the E1B 55 kD protein is not expressed are of great interest for the therapy of oncoses because the virus mutant replicates exclusively in tumor cells and not or to a negligible extent in primary normal cells (Bischoff et al., Science 274, 373-376, 1996; Kirn et al., Nature Med. 4, 1341-1342, 1998).
A preferred embodiment is the use of a permanent amniocytic cell line for producing adenovirus vectors, AAV (adeno-associated virus) vectors, retrovirus vectors, lentivirus vectors, chimeric adenovirus-AAV vectors, chimeric adenovirus-retrovirus vectors and/or chimeric adenovirus-lentivirus vectors. A use for producing herpes vectors is also possible.
AAV vectors normally comprise only the ITRs of AAV and some adjacent, noncoding AAV sequences. Their capacity for uptake of foreign DNA is about 4.5 kb. As described above, various systems exist for producing recombinant AAV vectors. It is common to all these systems that the components necessary for replication, expression and packaging of the recombinant vector are provided. Specifically, these comprise expression cassettes which code for the AAV rep and cap proteins, and the adenoviral helper functions. The adenoviral helper functions necessary for AAV production are the E1A, E1B, E2, E4 and VA genes. The E1A and E1B functions are provided in the E1A- and E1B-expressing amniocytic cell lines and can therefore be used to produce AAV vectors. The E2, E4 and VA functions can be provided by coinfection with adenovirus or by cotransfection with E2-, E4- and VA-expressing plasmids or by using adenovirus/AAV or herpes simplex virus/AAV hybrid vectors (Samulski et al., supra; Allen et al., supra; Tamayose et al., supra; Flotte et al., supra; Conway et al., supra; Chiorini et al., supra; Ferrari et al., supra; Salvetti et al., supra; Xiao et al., supra; Grimm et al., supra; Zhang et al., supra).
Retrovirus vectors, that is to say vectors derived from retroviruses, are likewise of great importance as vehicles for transfection within the scope of gene therapeutic procedures, for example for gene therapy in the central nervous system (Suhr et al., Arch. Neurol. 56, 287-292, 1999). Retroviral vectors can be produced in stable vector-producing cell lines or by transient transfection. The individual components used to produce retroviral vectors normally include one or more plasmids which express the structural proteins and the replication and integration proteins, as well as a plasmid which comprises the vector itself (Miller, in:. Retroviruses, Coffin, Hughes, Varmus ed., Cold Spring Harbor Laboratory Press, 1997, pp. 437-473). If those plasmids which contain an origin of replication, such as, for example, the SV40 origin of replication, are used, the amniocytic cell lines are modified so that proteins which promote replication of the plasmid are stably expressed. For example, in the case of plasmids which contain the SV40 origin of replication, an amniocytic cell line which expresses the T antigen of SV40 is used.
Lentivirus vectors are vectors derived from lentiviruses (Naldini et al., Science 272, 263-267, 1996; Dull et al., J. Virol. 72, 8463-8471, 1998). Lentiviral vectors can be produced in stable vector-producing cell lines or by transient transfection.
xe2x80x9cChimeric vectorsxe2x80x9d mean vectors which are the product of a fusion of nucleic acids from two or more different viral vectors. Permanent amniocytic cell lines can be used according to the present description for producing chimeric vectors. In this system, for example, an adenovirus vector, preferably an adenovirus vector of large capacity, harbors a DNA fragment which has the sequence information for an integrating virus which is derived, for example, from a retrovirus or from AAV. After transcription of a target cell, the integrating virus harboring a therapeutic gene is released from the adenoviral background (for example in the case of a retroviral insert by producing infectious retroviral particles which transduce neighboring cells and integrate stably as DNA). Examples of chimeric vectors produced in 293 cells have been described in the past, for example as chimeric adenovirus-retrovirus vectors (Feng et al., Nature Biotech. 15, 866-870, 1997) and as chimeric adenovirus-AAV vectors (Recchia et al., Proc. Natl. Acad. Sci. USA 96, 2615-2620, 1999). Production in E1A and E1B-expressing amniocytic cell lines is to be preferred because, in contrast to 293 cells, replication-competent vectors cannot be generated by homologous recombination.
Adenovirus vectors, that is to say vectors derived from adenoviruses, are of great importance in particular as vehicles for transfection within the scope of gene therapeutic procedures. The adenovirus vectors may be first-generation adenovirus vectors, second-generation adenovirus vectors, adenovirus vectors of large DNA capacity and/or deleted adenovirus vectors, which are produced with the aid of a permanent amniocytic cell line.
a) Production of First-generation Adenoviral Vectors
First-generation adenoviral vectors are usually characterized by deletions of the E1A and E1B genes. Some first-generation adenoviral vectors comprise, in addition to the deletion of the E1A and E1B genes, also deletions of the E3 region. E3 functions are dispensable for the growth of adenoviral vectors in cell culture.
First-generation adenoviral vectors can be produced in E1A- and E1B-expressing amniocytic cell lines. This is done by infecting the E1A- and E1B-expressing cells preferably with 3-5 infectious units per cell (3-5 MOI). After about 36 to 72 hours, the cells show a cytopathic effect. The cells are harvested by standard protocols. Adenoviral vector can be purified from them by CsCl density gradient centrifugation or by chromatographic processes.
b) Production of Second-generation Adenoviral Vectors
Second-generation adenoviral vectors are characterized by deletions of E1A and E1B genes. Some second-generation adenoviral vectors also comprise a deletion of the E3 region. In addition to the deletion of the E1A and E1B genes, second-generation adenoviral vectors are characterized by inactivation and preferably deletion of at least one other essential adenoviral gene, for example an E2A gene, an E2B gene and/or a E4 gene, or, for example, by deletions of E2 functions in combination with deletions of E4 functions.
To produce second-generation adenoviral vectors, the functions which the vector itself does not express, due to inactivation and/or deletion, must be provided by the amniocytic cell line. For this purpose, it is possible for amniocytic cell lines which stably express E1A and E1B to be stably modified by transfection of expression cassettes which express the gene products coding for one or more other adenoviral functions. For example, to produce a second-generation adenoviral vector which has, in addition to the deletion of the E1A and E1B genes, also a deletion of an E2A, E2B and/or E4 gene, the appropriate gene or genes is (are) introduced by transfection together with a selection marker into the E1A- and E1B-expressing amniocytic cell line. Cell clones which, in addition to the expression of E1A and E1B functions, also express E2A, E2B and/or E4 functions can then be used to produce the particular second-generation vector. The E2 and/or E4 genes are usually under the transcriptional control of a heterologous promoter, which either is constitutively active or can be regulated.
c) Production of Adenoviral Vectors of Large DNA Capacity
Adenoviral vectors of large DNA capacity are characterized by deletion of most or all of the viral coding sequences. These vectors preferably comprise only the viral ITRs and the viral packaging signal. The adenoviral functions are provided by a helper virus in trans. Various systems for producing adenoviral vectors of large DNA capacity have been described. It is common to all the systems described to date and using a helper virus that the helper virus corresponds to a replication-deficient, E1A- and E1B-deleted adenovirus. The helper virus comprises either a complete packaging signal (Mitani et al., Proc. Natl. Acad. Sci. USA 92, 3854-3858, 1995; Fisher et al., Virology 217, 11-22, 1996; Kumar-Singh and Chamberlain, Hum. Mol. Genet. 5, 913-921, 1996) or a mutated packaging signal (Kochanek et al., Proc. Natl. Acad. Sci. U.S.A. 93, 5731-5736, 1996). In the latter case, the vector is preferably packaged in viral capsids because the helper virus contains an attenuated packaging signal and therefore is packaged less efficiently. Alternatively, the packaging signal of the helper virus can be excised after the infection of the producer cell line by using a recombinase (Parks et al., Proc. Natl. Acad. Sci. USA 93, 13565-13570, 1996; Hardy et al., J. Virol. 71, 1842-1849, 1997). For example, the packaging signal of the helper virus can be flanked by loxP recognition sequences of bacteriophage P1. Expression of the Cre recombinase of bacteriophage P1 results in excision of the packaging signal of the helper virus. However, because of the absence of the packaging signal, no packaging of the helper virus into capsids takes place. The Cre recombinase gene of bacteriophage P1 is introduced by transfection together with a selection marker into the E1A- and E1B-expressing amniocytic cell line. Cell clones which, in addition to expression of E1A and E1B functions, also express the Cre function of bacteriophage P1 can then be used to produce the particular vector of large DNA capacity.
d) Production of xe2x80x9cDeletedxe2x80x9d Adenoviral Vectors
xe2x80x9cDeletedxe2x80x9d adenoviral vectors have been described as first-generation vectors which have loxP recognition sequences of bacteriophage P1 positioned in the viral genome in such a way that, on infection of Cre-expressing 293 cells, most of the viral coding sequences or all the viral coding sequences are deleted by recombination between the loxP recognition sequences. The genome size of these vectors is about 9 kb. The capacity for uptake of foreign DNA is likewise about 9 kb (Lieber et al., J. Virol, 70, 8944-8960, 1996). For use in the production of deleted adenoviral vectors, the Cre recombinase gene of bacteriophage P1 is introduced by transfection together with a selection marker into the E1A- and E1B-expressing amniocytic cell line. Cell clones which, in addition to expression of E1A and E1B functions, also express the Cre function of bacteriophage P1 can then be used to produce the particular deleted Ad vector.
e) Production of Tropism-modified Gene Transfer Vectors
In a preferred embodiment, the permanent amniocytic cell line is used to produce tropism-modified gene transfer vectors. The tropism of a virus and of a viral vector derived from this virus decides whether a particular cell type can be successfully transduced with a vector or not. Uptake of a gene transfer vector into a cell is the first step for successful gene transfer into this cell. The tropism of a viral vector is thus an essential factor for efficient in vitro or in vivo gene transfer into a particular cell or into a tissue. Interaction of the surface of a viral vector (of the capsid in the case of adenoviral or AAV vectors, of the virus envelope in the case of retroviral or lentiviral vectors) with the cell membrane of a target cell is necessary for uptake into a particular cell. Although the exact mechanism of uptake of a viral vector into a target cell sometimes varies between different vectors, in all cases the interaction of surface structures of the viral vector (usually protein ligands) with structures on the target cell (usually receptors or adhesion molecules) plays an essential part. Uptake of adenoviral vectors takes place, for example, by receptor-mediated endocytosis. This entails parts of the adenoviral capsid binding to cellular receptors. In the case of adenoviral vectors derived from Ad2 or Ad5, according to the current state of knowledge there is usually binding of part of the knob domain of the fiber proteins to the coxsackie adenovirus receptor (CAR) and part of the penton base to xcex1vxcex23 or xcex1vxcex25 integrins. The binding of the knob domain on CAR is, according to the current state of knowledge, necessary for adhesion of the vector to the cell membrane of the target cell, whereas binding of the penton base to integrins is necessary for internalization of the vector into the target cell.
Amniocytic cell lines can be used to produce tropism-modified vectors. This applies, for example, to the production of first- and second-generation adenoviral vectors, to adenoviral vectors of large DNA capacity, to deleted adenoviral vectors, to chimeric adenoviral vectors, to AAV vectors, to retroviral and/or lentiviral vectors. Various strategies can be used to produce tropism-modified vectors in amniocytic cell lines. The strategy used for the particular tropism modification may vary for different vectors (for example adenoviral vector, AAV vector, retroviral vector). It is common to the various strategies that the surface of the particular vector (virus capsid in the case of adenoviral and AAV vectors, virus envelope in the case of retroviral and lentiviral vectors) is altered so that the binding of the vector to the target cell is altered. Examples of modifications for adenoviral vectors are:
a) Exchange of fiber proteins between different serotypes: this results in adenoviral vectors whose capsid carries a fiber protein of a different serotype. Examples thereof are exchange of the natural fiber protein of adenoviral vectors derived from serotype 2 by a fiber protein derived from serotype 17 (Zabner et al., J. Virol. 73, 8689-8695, 1999) or from serotype 9 (Roelvink et al., J. Virol. 70, 7614-7621, 1996). Other examples are exchange of the natural fiber protein of adenoviral vectors derived from serotype 5 by a fiber protein derived from serotype 7a (Gall et al., J. Virol, 70, 2116-2123, 1996) or from serotype 3 (Stevenson et al., J. Virol. 71, 4782-4790, 1997; Krasnykh et al., J. Virol. 70, 6839-6846, 1996; Douglas et al., Neuromuscul. Disord. 7, 284-298, 1997).
b) Removal of the fiber protein: the fiber protein can be removed by processes of genetic manipulation so that uptake of the vector takes place solely via interaction of the penton base or of the hexon protein (Falgout et al., J. Virol. 62, 622-625, 1988; Legrand et al., J. Virol. 73, 907-919, 1999).
c) Modification of the C terminus of the fiber protein with a peptide: examples thereof are modification of the C terminus with a polylysine peptide (Yoshida et al., Hum. Gene Ther. 9, 2503-2515, 1998: Wickham et al., Nat. Biotechnol. 14, 1570-1573, 1996; Wickham et al., J. Virol. 71, 8221-8229, 1997), a polyhistidine peptide (Douglas et al., Nat. Biotechnol. 17, 470-475, 1999) or a gastrin-releasing peptide (Michael et al., Gene Ther. 2, 660-668, 1995).
d) Modification of parts of the knob domain of the fiber protein by insertion of a peptide: examples thereof are insertion of a FLAG epitope (Krasnykh et al., J. Virol. 72, 1844-1852, 1998) or insertion of an RGD peptide (Dmitriev et al., J. Virol. 72, 9706-9713, 1998; Kasono et al., Clin Cancer Res. 5, 2571-2579, 1999).
e) Modification of the penton base: one example thereof is replacement of an RGD motif within the penton base by an LDV motif with the aim of mediating binding of the vector to xcex14xcex21 integrins (Wickham et al., Gene Ther. 2, 750-756, 1995).
f) Modification of the hexon protein: one example thereof is insertion of an epitope derived from poliovirus type 3 (Crompton et al., J. Gen. Virol. 75, 133-139, 1994).
An alternative strategy which can be used to alter the tropism of vectors produced in amniocytic cell lines is based on the use of ligands which mediate binding of the vector to cell membrane structures such as, for example, cellular receptors or adhesion molecules. These ligands may be peptides, proteins or else antibodies. The ligands can be linked to the surface of the vectors by various processes. The linkage of the ligands to the surface of the vectors (of the capsids in the case of adenoviral or AAV vectors) can be produced by using antibodies or by a chemical crosslinking reaction. On use of antibodies it is possible to use antibodies whose specificity is directed against the capsid of the vector (for example against the knob domain of the fiber protein). Alternatively, it is possible to use antibodies whose specificity is directed against an epitope which has been introduced as neoepitope (for example a FLAG epitope or a myc epitope) into the capsid of the vector. Examples thereof are well known to the skilled worker. Examples of the use of bispecific antibodies are described in Wickham et al., J. Virol. 70, 6831-6838, 1996 (anti-FLAG/anti-xcex1-integrin); in Wickham et al., Cancer Immunol. Immunther. 45, 149-151, 1997; Harari et al., Gene Ther. 6, 801-807, 1999 (anti-FLAG/anti-E-selectin) for transduction of endothelial cells; in Miller et al., Cancer Res. 58, 5738-5748, 1998; Blackwell et al., Arch. Otolaryngol. Head Neck Surg. 125, 856-863, 1999 (anti-Ad/anti-EGFR) for transduction of tumor cells; in Wickham et al., J. Virol. 71, 7663-7669, 1997 (anti-FLAG/anti-CD3) for transduction of T cells; in Tillman et al., J. Immunol. 162, 6378-6383, 1999 (anti-CD40/anti-Ad) for transduction of dendritic cells. Examples of the use of single-chain antibodies with specificity for one virus capsid determinant which is coupled to a ligand are described in Watkins et al., Gene Ther. 4, 1004-1012, 1997; in Goldman et al., Cancer Res. 57, 1447-1451, 1997; Rancourt et al., Clin. Cancer Res. 4, 2455-2461, 1998; Gu et al., Cancer Res. 59, 2608-2614, 1999; Rogers et al., Gene Ther. 4, 1387-1392, 1997 (anti-Ad/FGF2) for transduction of FGF2-receptor-expressing tumor cells; in Douglas et al., Nat. Biotechnol. 14, 1574-1578, 1996; Douglas et al., Neuromuscular Disord. 7, 284-298, 1997 (anti-Ad/Folat) for transduction of tumor cells which express the folic acid receptor on the cell surface.
In the case of gene transfer vectors in which the natural tropism has been abolished and replaced by another tropism, for example by introducing a ligand into the knob domain of the fiber protein of Ad5, it may be necessary to modify a permanent amniocytic cell line by the preferably stable expression of a receptor which recognizes this new ligand (Douglas et al., Nat. Biotechnol. 17, 470-475, 1999). It is likewise possible for the permanent amniocytic cell line to be used to produce gene transfer vectors which have a defect in the production of one or more structural proteins. This is done by complementing the particular defects of the gene transfer vector in the permanent amniocytic cell line. For example, an adenoviral vector which has a mutation in the gene coding for the fiber protein can be produced in an amniocytic cell line which complements the defect in the fiber protein. This is achieved by introducing a fiber expression cassette into the amniocytic cell line and stable or inducible expression of the fiber protein in this amniocytic cell line (Von Seggern et al., J. Gen. Virol. 79, 1461-1468, 1998). The fiber protein expressed in the amniocytic cell line may be a natural, unmodified fiber protein or else an altered, for example tropism-modified, fiber protein (Von Seggern et al., supra). It is also possible to produce adenoviral vectors completely lacking the fiber protein in the permanent amniocytic cell line (Legrand et al., J. Virol., 73, 907-919, 1999; Von Seggern et al., J. Virol. 73, 1601-1608, 1999).
The use of E1A- and E1B-expressing amniocytic cell lines is to be preferred because, in contrast to 293 cells, no generation of replication-competent vectors can take place by homologous recombination. In a particular embodiment of the aspect of the use of an amniocytic cell line for producing gene transfer vectors, this cell line is the cell line according to the invention.
Therapeutic Genes
The products of the genes, in particular of the therapeutic genes, which can be encoded and expressed by vectors produced in transformed amniotic cells, that is to say a permanent amniotic cell line, can be, for example, any muscle proteins, coagulation factors, membrane proteins or cell cycle proteins. Examples of proteins which can be expressed by vectors produced in transformed amniocytes are dystrophin (Hoffman et al., Cell 51, 919, 1987), factor VIII (Wion et al., Nature 317, 726 1985), cystic fibrosis transmembrane regulator protein (CFTR) (Anderson et al., Science 251, 679, 1991), ornithine transcarbamylase (OTC) (Murakami et al., J. Biol. Chem., 263, 18437, 1988), alphal-antitrypsin (Fagerhol et al., in: Hum. Genet., vol. 11, Harris ed., Plenum, New York, p. 1, 1981). The genes coding for proteins are known and can be cloned from genomic or cDNA banks. Examples of such genes are the dystrophin gene (Lee et al., Nature 349, 334, 1991), the factor VIII gene (Toole et al., Nature 312, 342 1984), the CFTR gene (Rommens et al., Science 245, 1059, 1989, Riordan et al., Science 245, 1066, 1989), the OTC gene (Horwich et al., Science 224, 1066, 1984), and the alphal-antitrypsin gene (Lemarchand et al., Proc. Natl. Acad. Sci. USA, 89, 6482, 1992).
Examples of other genes expressed by vectors which can be produced in transformed amniocytes are the p53 gene for treating oncoses (Wills et al., Hum. Gene Ther. 5, 1079, 1994, Clayman et al., Cancer Res. 55, 1, 1995), the Rb gene for treating vascular proliferative disorders (Chang et al., Science 267, 518, 1995), or the thymidine kinase gene of herpes simplex virus (HSV) type 1 for the therapy of oncoses. The gene expressed by vectors produced in transformed amniocytes does not necessarily code for a protein. Thus, for example, it is possible for functional RNAs to be expressed. Examples of such RNAs are antisense RNAs (Magrath, Ann. Oncol. 5, Suppl 1), 67-70 1994, Milligan et al., Ann. NY Acad. Sci. 716, 228-241, 1994, Schreier, Pharma. Acta. Helv., 68, 145-159 1994), and catalytic RNAs (Cech, Biochem. Soc. Trans. 21, 229-234, 1993; Cech, Gene 135, 33-36, 1993; Long et al., FASEB J. 7, 25-30, 1993; Rosi et al., Pharm. Therap. 50, 245-254, 1991).
Vectors produced in transformed amniocytes may, in addition to the therapeutic gene, comprise any reporter gene in order to be able to follow expression of the vector better. Examples of reporter genes are known in the prior art and include, for example, the xcex2-galactosidase gene (Fowler et al., Proc. Natl. Acad. Sci. USA 74, 1507, 1977).
Vectors which can be produced in transformed amniocytes may comprise more than a single gene. The maximum number of genes which can be produced in such vectors depends on the uptake capacity of the particular vector and on the size of the genes.
The choice of the promoters which control expression of the therapeutic genes of vectors produced in transformed amniocytes is not critical. Viral or nonviral promoters which show constitutive, tissue-specific or regulable activity can be used for expressing a protein or a functional RNA. The SV40 or cytomegalovirus promoter (Andersson et al., J. Biol. Chem. 264, 8222-8229, 1964) can be used, for example, for constitutive expression of a gene. The use of the muscle creatine kinase (MCK) promoter permits tissue-specific expression of a protein or of a functional RNA in skeletal muscle and myocardium. Gene expression can be controlled quantitatively and qualitatively by the use of a regulable system (Furth et al., Proc. Natl. Acad. Sci. USA 91, 9302-9306, 1994).
It is possible to include in vectors which can be produced in transformed amniocytes genetic elements which influence the behavior of the vector inside the recipient cell. Examples of such elements are elements which facilitate nuclear targeting of the vector DNA (Hodgson, Biotechnology 13, 222-225, 1995).
Vectors produced in this way can be used in vitro or in vivo. An in vitro gene transfer takes place outside the body, for example by adding the vector to cells in culture or to primary cells which have been taken from the body for the purpose of gene transfer. In the case of in vivo gene transfer, vector particles can be applied in various ways depending on the tissue which is to be transduced. Examples are injection into the arterial or venous vascular system, direct injection into the relevant tissue (for example liver, brain, muscle), instillation into the relevant organ (for example lung or gastrointestinal tract) or direct application onto a surface (for example skin or bladder).
The following figures and example are intended to illustrate the invention in detail without restricting it thereto.